A multiphoton microscopy such as two-photon excitation fluorescence (2PEF) microscopy is a fluorescence imaging technique that is based on nonlinear optical absorption in fluorescently-tagged structures and is useful for visualizing fluorescently-tagged cells in living animal tissue. The nonlinear excitation involves absorption of two or more excitation photons to excite a single fluorescent molecule from a low energy ground state to a higher energy state. Once the fluorophore is in an excited state, the fluorophore decays to the ground energy state to release energy in the form of single-photon fluorescence. In two-photon absorption, the fluorescence signal intensity scales as the square of the laser excitation intensity and a high photon flux in a volume is required. The 2PEF technique can be used to overcome tissue scattering through inherent optical sectioning by generating in-focus images of a plane in a three-dimensional (3D) sample, without requiring physical sectioning of the sample. In wide-field fluorescence microscopy, the entire field of view (FOV) is illuminated by an ultraviolet (UV) source which generates fluorescence throughout the sample. The sample is “imaged” with a microscope objective that replicates the image on a viewer's retina or a camera. If the sample is too thick (i.e. more than a few cells thick), fluorescence is generated everywhere structures are labeled, and an acquired image may contain signals from tagged structures at varying depths to appear as a blurry image.
2PEF provides optical sectioning through nonlinear excitation of fluorophores which tends to occur in the small, sub-micrometer focus of the microscope objective. Because out-of-plane fluorescence is not generated in the first place, detected photons are mostly attributed to the current location of the focal volume. This optical sectioning in 2PEF can be paired with a line-scanning microscope to generate 3D images of fluorescent structures.